Influence of caffeine and hyaluronic acid on collagen biosynthesis in human skin fibroblasts

Abstract

Aim: The aim of this study was to evaluate the effect of caffeine on collagen biosynthesis in human skin fibroblasts and the influence of hyaluronic acid (HA) on this process.

Materials and methods: Collagen, [(3)H]-thymidine incorporation, and prolidase activity were measured in confluent human skin fibroblast cultures that had been treated with 1, 2, and 5 mM caffeine and with caffeine and 500 μg/mL HA. Western immunoblot analysis was performed to evaluate expression of β1-integrin receptor, insulin-like growth factor receptor phospho-Akt protein and mitogen-activated protein kinase (phospho-extracellular signal-regulated kinase).

Results: Caffeine inhibited collagen biosynthesis in a dose-dependent manner. The mechanism of this process was found at the level of prolidase activity. Caffeine significantly inhibited the enzyme activity. The addition of HA had no effect on collagen biosynthesis or prolidase activity in fibroblasts incubated with caffeine. Caffeine also had an inhibitory effect on DNA biosynthesis. HA, however, did not have any significant effect on this process. The inhibition of the expression of β1-integrin and insulin-like growth factor receptor in fibroblasts incubated with the caffeine indicates a possible mechanism of inhibition of collagen biosynthesis.

Conclusion: Caffeine reduces collagen synthesis in human cultured skin fibroblasts. HA did not have any significant protective effect on this process. This is the first study to our knowledge that reports caffeine-induced inhibition of collagen synthesis in human skin fibroblasts.

Keywords: caffeine; collagen; fibroblast; hyaluronic acid.

Figure 1

Collagen biosynthesis measured as 5[³H]-proline incorporation into proteins susceptible to the action of bacterial collagenase in confluent human skin fibroblasts incubated for 24 hours with different concentrations of caffeine and hyaluronic acid (HA). Note: Error bars represent ± standard deviation; n=3.

Figure 2

Prolidase activity in confluent human skin fibroblasts incubated for 24 hours with different concentrations of caffeine and hyaluronic acid (HA). Note: Error bars represent ± standard deviation; n=3.

Figure 3

DNA biosynthesis measured as [³H]-thymidine incorporation into DNA in human skin fibroblasts (control) incubated for 24 hours with different concentrations of caffeine and hyaluronic acid (HA). Note: Error bars represent ± standard deviation; n=3.

Figure 4

Western immunoblot analysis for β₁-integrin (A), insulin-like growth factor I receptor (B), extracellular signal-regulated kinase (ERK1/2) (C), Akt kinase (D), and β-actin (E) in subconfluent human skin fibroblasts (control), cells treated with or without hyaluronic acid for 24 hours. Notes: 1: control, 2: caffeine 1 mM, 3: caffeine 2 mM, 4: caffeine 1 mM, 5: hyaluronic acid, 6: caffeine 1 mM and hyaluronic acid, 7: caffeine 2 mM and hyaluronic acid; 8: caffeine 5 mM and hyaluronic acid. Samples used for electrophoresis consisted of 20 μg protein of pooled cell extracts (8). The intensity of the bands staining was quantified by densitometry analysis.